Detection of Human Immunodeficiency Virus type 1

ABSTRACT

Amplification oligonucleotides and hybridization assay probes which distinguish Human Immunodeficiency Virus type 1 from other viruses.

[0001] This application is a continuation in part of Kacian and Fultz, entitled “Nucleic Acid Sequence Amplification Methods,” U.S. Ser. No. 07/379,501, filed Jul. 11, 1989, and Kacian and Fultz, entitled “Nucleic Acid Sequence Amplification Methods,” U.S. Ser. No. 07/550,837, filed Jul. 10, 1990, both hereby incorporated by reference herein.

FIELD OF THE INVENTION

[0002] This invention relates to the design and construction of amplification oligonucleotides and probes to Human Immunodeficiency Virus Type 1 (HIV), which allow detection of the organism in a test sample.

BACKGROUND OF THE INVENTION

[0003] This section provides a brief outline of relevant areas. None of the art cited or referred to is admitted to be prior art to the claims. Laboratory diagnosis of Human Immunodeficiency Virus Type 1 in humans is currently performed by demonstration of the presence of viral antigen (p24) or anti-HIV-1 antibodies in serum. Direct detection of viral DNA, however, is a more useful diagnostic tool in some populations, such as infants born to seropositive mothers. Detection of viral DNA is more rapid and less hazardous than culture. Direct hybridization lacks adequate sensitivity in most patients (Shaw et al. Science 226:1165-1171, 1984). Many references mention oligonucleotides said to have use in detection of Human Immunodeficiency Virus. Most of these references also mention the use of polymerase chain reaction (PCR). These references include the following: Kwok et al., J. Virol. 61: 1690-1694, 1987; Agius et al., J. Virol. Meth., 30:141-150, 1990; Albert and Fenyo, J. Clin. Microbiol. 28:1560-1564, 1990; Bell and Ratner, AIDS Res. and Human Retroviruses 5:87-95, 1989; Bruisten et al., Vox Sang 61:24-29, 1991; Clarke et al., AIDS 4:1133-1136, 1990; Coutlee et al., Anal. Biochem. 181:96-105, 1989; Dahlen et al., J. Clin. Microbiol. 29:798-804, 1991; Dudding et al., Biochem. Biophys. Res. Comm. 167:244-250, 1990; Ferrer-Le-Coeur et al., Thrombosis and Haemostasis 65:478-482, 1991; Goswami et al., AIDS 5:797-803, 1991; Grankvist et al., AIDS 5:575-578, 1991; Guatelli et al., J. Virol. 64:4093-4098, 1990; Hart et al., Lancet 2 (8611):596-599, 1988; Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7280, 1991; Keller et al., Anal. Biochem. 177:27-32, 1989; Kumar et al., AIDS Res. and Human Retroviruses 5:345-354, 1989; Linz et al., J. Clin. Chem. Clin. Biochem. 28:5-13, 1990; Mano and Chermann, Res. Virol. 142:95-104, 1991; Mariotti et al., AIDS 4:633-637, 1990; Mariotti et al., Transfusion 30:704-706, 1990; Meyerhans et al., Cell 58:901-910, 1989; Mousset et al., AIDS 4:1225-1230, 1990; Ou et al., Science 239:295-297, 1988; Pang et al., Nature 343:85-89, 1990; Paterlini et al., J. Med. Virol. 30:53-57, 1990; Perrin et al., Blood 76:641-645, 1990; Preston et al., J. Virol. Meth. 33:383-390, 1991; Pritchard and Stefano, Ann. Biol. Clin. 48:492-497, 1990; Rudin et al., Eur. J. Clin. Microbiol. Infect. Dis. 10:146-156, 1991; Shoebridge et al., AIDS 5:221-224, 1991; Stevenson et al., J. Virol. 64:3792-3803, 1990; Truckenmiller et al., Res. Immunol. 140:527-544, 1989; Van de Perre, et al., New Eng. J. Med. 325:593-598, 1991; Varas et al., BioTechniques 11:384-391, 1991; Velpandi et al., J. Virol. 65:4847-4852, 1991; Williams et al., AIDS 4:393-398, 1990; Zachar et al., J. Virol. Meth. 33:391-395, 1991; Zack et al. Cell 61:213-222, 1990; Findlay et al., entitled “Nucleic acid test article and its use to detect a predetermined nucleic acid,” PCT/US90/00452; Gingeras et al., entitled “Nucleic acid probe assay methods and compositions,” PCT/US87/01966; Brakel and Spadoro, entitled “Amplification capture assay,” EPO application number 90124738.7, publication number 0 435 150 A2; Moncany and Montagnier, entitled “Séquences nucléotidiques issues du génome des rétrovirus du typ hiv-1, hiv-2 et siv, et leurs applications notamment pour 1′amplification des génomes de ces rétrovirus et pour le diagnostic in-vitro des infections dues á ces virus,” EPO application number 90401520.3, publication number 0 403 333 A2; Urdea, entitled “DNA-dependent RNA polymerase transcripts as reporter molecules for signal amplification in nucleic acid hybridization assays,” PCT/US91/00213; Musso et al., entitled “Lanthanide chelate-tagged nucleic acid probes,” PCT/US88/03735; Chang, entitled “Cloning and expression of HTLV-III DNA,” EPO application number 85307260.1, publication number 0 185 444 A2; and Levenson, entitled “Diagnostic kit and method using a solid phase capture means for detecting nucleic acids,” EPO application number 89311862.0, publication number 0 370 694; and Sninsky et al., U.S. Pat. No. 5,008,182.

SUMMARY OF THE INVENTION

[0004] This invention discloses novel amplification oligonucleotides and detection probes for the detection of Human Immunodeficiency Virus Type 1. The probes are capable of distinguishing between the Human Immunodeficiency Virus type 1 and its known closest phylogenetic neighbors. The amplification oligonucleotides and probes may be used in an assay for the detection and/or quantitation of Human Immunodeficiency Virus nucleic acid.

[0005] It is known that a nucleic acid sequence able to hybridize to a nucleic acid sequence sought to be detected (“target sequence”) can serve as a probe for the target sequence. The probe may be labelled with a detectable moiety such as a radioisotope, antigen or chemiluminescent moiety to facilitate detection of the target sequence. A background description of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences is provided in Kohne, U.S. Pat. No. 4,851,330, and Hogan et al., EPO Patent application No. PCT/US87/03009, entitled “Nucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organisms.”

[0006] It is also known that hybridization may occur between complementary nucleic acid strands including; DNA/DNA, DNA/RNA, and RNA/RNA. Two single strands of deoxyribo-(“DNA”) or ribo- (“RNA”) nucleic acid, formed from nucleotides (including the bases adenine (A), cytosine (C), thymidine (T), guanine (G), uracil (U), or inosine (I)), may hybridize to form a double-stranded structure in which the two strands are held together by hydrogen bonds between pairs of complementary bases. Generally, A is hydrogen bonded to T or U, while G is hydrogen bonded to C. At any point along the hybridized strands, therefore, one may find the classical base pairs AT or AU, TA or UA, GC, or CG. Thus, when a first single strand of nucleic acid contains sufficient contiguous complementary bases to a second, and those two strands are brought together under conditions which will promote their hybridization, double-stranded nucleic acid will result. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may be formed. The present invention includes the use of probes or primers containing nucleotides differing in the sugar moiety, or otherwise chemically modified, which are able to hydrogen bond along the lines described above.

[0007] Thus, in a first aspect, the invention features hybridization assay probes able to distinguish Human Immunodeficiency Virus type 1 from other viruses found in human blood or tissues, and amplification oligonucleotides able to selectively amplify Human Immunodeficiency Virus nucleic acid. Specifically, the probes are nucleotide polymers which hybridize to the nucleic acid region of Human Immunodeficiency Virus type 1 corresponding to bases 763-793 of HIV type 1, (HXB2 isolate GenBank accession number K03455), or any of the regions corresponding to bases 1271-1301, 1358-1387, 1464-1489, 1501-1540, 1813-1845, 2969-2999, 3125-3161, 4148-4170, 4804-4832, 5950-5978, 9496-9523, 510-542, and 624-651; preferably, the oligonucleotide comprises, consists essentially of, or consists of the sequence (reading 5′ to 3′ ) (SEQ ID GACTAGCGGAGGCTAGAAGGAGAGAGATGGG NO: 1) (SEQ ID GAAGGCTTTCAGCCCAGAAGTAATACCCATG NO: 2) (SEQ ID ATTTGCATGGCTGCTTGATGTCCCCCCACT NO: 3) (SEQ ID CTTCCCCTTGGTTCTCTCATCTGGCC NO: 4) (SEQ ID GTCATCCATCCTATTTGTTCCTGAAGGGTACTAGTAG NO: 5) (SEQ ID CTCCCTGACATGCTGTCATCATTTCTTCTAGTG NO: 6) (SEQ ID GTGGAAGCACATTGTACTGATATCTAATCCC NO: 7) (SEQ ID GCTCCTCTATTTTTGTTCTATGCTGCCCTATTTCTAA NO: 8) (SEQ ID CCTTTGTGTGCTGGTACCCATGC NO: 9) (SEQ ID CTACTATTCTTTCCCCTGCACTGTACCCC NO:10) (SEQ ID AAAGCCTTAGGCATCTCCTATGGCAGGAA NO:11) (SEQ ID GCAGCTGCTTATATGCAGGATCTGACGG NO:12) (SEQ ID CAAGGCAAGCTTTATTGAGGCTTAAGCAGTGGG NO:13) (SEQ ID ATCTCTAGCAGTGGCGCCCGAACACGGA NO:14)

[0008] or RNA equivalents thereto (SEQ. ID. Nos. 67-80), or oligonucleotides complementary thereto (SEQ. ID. Nos. 53-66), or RNA equivalents to the oligonucleotides complementary thereto (SEQ. ID. Nos. 81-94).

[0009] The oligonucleotides are used with or without a helper probe as described below. The use of helper probes (e.g., SEQ. ID. Nos. 15-18) and complementary oligonucleotides to the helper probes (e.g., SEQ. ID. Nos. 95-98) and RNA equivalents thereto (e.g., SEQ. ID. Nos. 132-140) enhances nucleic acid hybridization.

[0010] By “consists essentially of” is meant that the probe is provided as a purified nucleic acid which under stringent hybridizing conditions hybridizes with the target sequence and not with other related target sequences present in either other virus nucleic acids or human nucleic acids. Such a probe may be linked to other nucleic acids which do not affect such hybridization. Generally, it is preferred that the probe is between 15 to 100 (most preferably between 20 and 50) bases in size. It may, however, be provided in a vector.

[0011] In a related aspect, the invention features the formation of nucleic acid hybrids formed by the hybridization of the probes of this invention with target nucleic acid under stringent hybridization conditions. Stringent hybridization conditions involve the use 0.05 M lithium succinate buffer containing 0.6 M LiCl at 60° C. The hybrids are useful because they allow the specific detection of viral nucleic acid.

[0012] In another related aspect, the invention features amplification oligonucleotides useful for specific detection of Human Immunodeficiency Virus type 1 in an amplification assay. The amplification oligonucleotides are complementary to conserved regions of HIV genomic nucleic acid and are nucleotide polymers able to hybridize to regions of the nucleic acid of HIV corresponding to HIV-1 HXB2R bases 682-705, 800-822, 1307-1337, 1306-1330, 1315-1340, 1395-1425, 1510-1535, 1549-1572, 1743-1771, 1972-1989, 2868-2889, 3008-3042, 3092-3124, 3209-3235, 4052-4079, 4176-4209, 4169-4206, 4394-4428, 4756-4778, 4835-4857, 4952-4969, 5834-5860, 5979-5999, 9431-9457, 9529-9555, 449-473, 550-577, 578-601, 579-600, 624-646, and 680-703.

[0013] Specifically, such amplification oligonucleotides consist, comprise, or consist essentially of those selected from (reading 5′ to 3′ ): (X) CTCGACGCAGGACTCGGCTTGCTG (SEQ. ID. NO. 19), (X) CTCCCCCGCTTAATACTGACGCT (SEQ. ID. NO. 20), (X) GGCAAATGGTACATCAGGCCATATCACCTAG (SEQ. ID. NO. 21), (X) GGGGTGGCTCCTTCTGATAATGCTG (SEQ. ID. NO. 22), (X) CAGAACGAGCCACCCCACAAGATTTA (SEQ. ID. NO. 23), (X) GACCATCAATGAGGAAGCTGCAGAATG (SEQ. ID. NO. 24), (X) CCCATTCTGCAGCTTCCTCATTGAT (SEQ. ID. NO. 25), (X) AGTGACATAGCAGGAACTA (SEQ. ID. NO. 26), (X) CCATCCTATTTGTTCCTGAAGGGTAC (SEQ. ID. NO. 27), (X) AGATTTCTCCTACTGGGATAGGT (SEQ. ID. NO. 28), (X) GAAACCTTGTTGAGTCCAAAATGCGAACCC (SEQ. ID. NO. 29), (X) TGTGCCCTTCTTTGCCAC (SEQ. ID. NO. 30), (X) CAGTACTGGATGTGGGTGATGC (SEQ. ID. NO. 31), (X) GTCATGCTACTTTGGAATATTTCTGGTGATCCTTT (SEQ. ID. NO. 32), (X) CAATACATGGATGATTTGTATGTAGGATCTGAC (SEQ. ID. NO. 33), (X) ACCAAAGGAATGGAGGTTCTTTCTGATG (SEQ. ID. NO. 34), (X) GCATTAGGAATCATTCAAGCACAACCAG (SEQ. ID. NO. 35), (X) GCACTGACTAATTTATCTACTTGTTCATTTCCTC (SEQ. ID. NO. 36), (X) GGGATTGGAGGAAATGAACAAGTAGATAAATTAGTCAG (SEQ. ID. NO. 37), (X) TGTGTACAATCTAGTTGCCATATTCCTGGACTACA (SEQ. ID. NO. 38), (X) CAAATGGCAGTATTCATCCACA (SEQ. ID. NO. 39), (X) GTTTGTATGTCTGTTGCTATTAT (SEQ. ID. NO. 40), (X) CCCTTCACCTTTCCAGAG (SEQ. ID. NO. 41), (X) GAGCCCTGGAAGCATCCAGGAAGTCAG (SEQ. ID. NO. 42), (X) CTTCGTCGCTGTCTCCGCTTC (SEQ. ID. NO. 43), (X) CAAGGGACTTTCCGCTGGGGACTTTCC (SEQ. ID. NO. 44), (X) GTCTAACCAGAGAGACCCAGTACAGGC (SEQ. ID. NO. 45), (X) GTACTGGGTCTCTCTGGTTAGACCA (SEQ. ID. NO. 46), (X) CACACAACAGACGGGCACACACTACTTG (SEQ. ID. NO. 47), (X) CTGAGGGATCTCTAGTTACCAGAGT (SEQ. ID. NO. 48), (X) CTCTGGTAACTAGAGATCCCTCA (SEQ. ID. NO. 49), (X) GTTCGGGCGCCACTGCTAGAGAT (SEQ. ID. NO. 50), (X) GCAAGCCGAGTCCTGCGTCGAGA (SEQ. ID. NO. 51)

[0014] and the RNA equivalents thereto (SEQ. ID. Nos. 99-131). Where (X) is nothing or a 5′ oligonucleotide sequence that is recognized by an enzyme, including but not limited to the promoter sequence for T7, T3, or SP6 RNA polymerase, which enhances initiation or elongation of RNA transcription by an RNA polymerase. One example of X includes the sequence SEQ. ID. NO. 52: 5′-AATTTAATACGACTCACTATAGGGAGA-3′.

[0015] These amplification oligonucleotides are used in a nucleic acid amplification assay such as the polymerase chain reaction or an amplification reaction using RNA polymerase, DNA polymerase and RNase H or its equivalent, as described by Kacian and Fultz, supra, and by Sninsky et al. U.S. Pat. No. 5,079,351, both hereby incorporated by reference herein.

[0016] The amplification oligonucleotides and probes of this invention offer a rapid, non-subjective method of identification and quantitation of a sample for specific sequences unique to strains of Human Immunodeficiency Virus type 1.

[0017] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] We have discovered particularly useful DNA probes complementary to particular nucleic acid sequences of Human Immunodeficiency Virus type 1. Furthermore, we have successfully used these probes in a specific assay for the detection of Human Immunodeficiency Virus type 1, distinguishing it from the known and presumably most closely related taxonomic or phylogenetic neighbors found in human blood or tissues.

[0019] We have also identified particularly useful amplification oligonucleotides which are complementary to the Human Immunodeficiency Virus type 1 nucleic acid, and have used these oligonucleotides, e.g., as primers or promoter primer combinations (i.e., a primer having a promoter sequence attached), to amplify the nucleic acid of Human Immunodeficiency Virus, allowing its direct detection in a sample.

[0020] Useful guidelines for designing amplification oligonucleotides and probes with desired characteristics are described herein. The optimal sites for amplifying and probing contain two, and preferably three, conserved regions greater than about 15 bases in length, within about 350 bases, and preferably within 150 bases, of contiguous sequence. The degree of amplification observed with a set of primers or promotor/primers depends on several factors, including the ability of the oligonucleotides to hybridize to their complementary sequences and their ability to be extended enzymatically. Because the extent and specificity of hybridization reactions are affected by a number of factors, manipulation of those factors will determine the exact sensitivity and specificity of a particular oligonucleotide, whether perfectly complementary to its target or not. The importance and effect of various assay conditions are known to those skilled in the art as described in Hogan et al., EPO Patent Application No. PCT/US87/03009, entitled “Nucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organisms”; and Milliman, entitled “Nucleic Acid Probes to Haemophilus influenzae,” U.S. Ser. No. 07/690,788, filed Apr. 25, 1991 assigned to the same assignee as the present application and hereby incorporated by reference herein.

[0021] The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly homologous base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, oligonucleotide probes preferred in this invention are between about 10 to 50 bases in length and are sufficiently homologous to the target nucleic acid to hybridize under stringent hybridization conditions. We have found that optimal primers have target-binding regions of 18-38 bases, with a predicted Tm (melting temperature) to target of about 65° C.

[0022] Amplification oligonucleotides or probes should be positioned so as to minimize the stability of the oligomer:nontarget (i.e., nucleic acid with similar sequence to target nucleic acid) nucleic acid hybrid. It is preferred that the amplification oligomers and detection probes are able to distinguish between target and non-target sequences. In designing probes, the differences in these Tm values should be as large as possible (e.g., at least 2° C. and preferably 5° C.).

[0023] Regions of the nucleic acid which are known to form strong internal structures inhibitory to hybridization are less preferred. Examples of such structures include hairpin loops. Likewise, probes with extensive self-complementarity should be avoided.

[0024] The degree of non-specific extension (primer-dimer or non-target copying) can also affect amplification efficiency, therefore primers are selected to have low self- or cross- complementarity, particularly at the 3′ ends of the sequence. Long homopolymer tracts and high GC content are avoided to reduce spurious primer extension. Commercial computer programs are available to aid in this aspect of the design. Available computer programs include MacDNASIS™ 2.0 (Hitachi Software Engineering American Ltd.) and OLIGO ver. 4.1 (National Bioscience).

[0025] Hybridization is the association of two single strands of complementary nucleic acid to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. If the target is an integrated genomic sequence then it will naturally occur in a double stranded form, as is the case with the product of the polymerase chain reaction (PCR). These double-stranded targets are naturally inhibitory to hybridization with a probe and require denaturation prior to the hybridization step. Finally, there can be intramolecular and intermolecular hybrids formed within a probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. Commercial computer programs are available to search for this type of interaction. Available computer programs include MacDNASIS™ 2.0 (Hitachi Software Engineering American Ltd.) and OLIGO ver. 4.1 (National Bioscience).

[0026] Once synthesized, selected oligonucleotide probes may be labelled by any of several well known methods. 2 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning 11 (2d ed. 1989). Useful labels include radio-isotopes as well as non-radioactive reporting groups. We currently prefer to use acridinium esters.

[0027] Oligonucleotide/target hybrid melting temperature may be determined by isotopic methods well known to those skilled in the art. It should be noted that the Tm for a given hybrid will vary depending on the hybridization solution being used. Sambrook, et al. supra.

[0028] Rate of hybridization may be measured by determining the C_(o)t_(½). The rate at which a probe hybridizes to its target is a measure of the thermal stability of the target secondary structure in the probe region. The standard measurement of hybridization rate is the C_(o)t_(½) which is measured as moles of nucleotide per liter times seconds. Thus, it is the concentration of probe times the time at which 50% of maximal hybridization occurs at that concentration. This value is determined by hybridizing various amounts of probe to a constant amount of target for a fixed time. The C_(o)t_(½) is found graphically by standard procedure.

[0029] The following examples set forth oligonucleotide probes complementary to a unique nucleic acid sequence from a target organism, and their use in a hybridization assay.

EXAMPLES

[0030] Probes specific for Human Immunodeficiency Virus type 1 were identified by comparison of sequences obtained from the published database GenBank. Sequences ID Nos. 1-12 were characterized and shown to be specific for Human Immunodeficiency Virus type 1. Phylogenetically near neighbors including Human Immunodeficiency Virus type 2, Human T-cell Leukemia Virus type 1 and Human T-Cell Leukemia Virus type 2 were used as comparisons with the sequence of Human Immunodeficiency Virus Type 1.

Example 1

[0031] Probes for HIV

[0032] A hybridization protection assay was used to demonstrate the reactivity and specificity of the probes for Human Immunodeficiency Virus type 1. The probes were first synthesized with a non-nucleotide linker, then labelled with a chemiluminescent acridinium ester (AE) as described by Arnold, et al., PCT/US88/03361, entitled “Acridinium Ester Labeling and Purification of Nucleotide Probes,” hereby incorporated by reference herein. The acridinium ester attached to an unhybridized probe is susceptible to hydrolysis and rendered non-chemiluminescent under mild alkaline conditions. However, the acridinium ester attached to hybridized probe is relatively resistant to hydrolysis. Thus, it is possible to assay for hybridization of acridinium ester-labelled probe by incubation with an alkaline buffer, followed by detection of chemiluminescence in a luminometer. Results are given in Relative Light Units (RLU); the quantity of photons emitted by the labelled-probe measured by the luminometer.

[0033] In the following experiment, DNA prepared from clones containing full or partial sequences of the target viruses was assayed. An example of a method for preparing the DNA from clones is provided by Sambrook et al, supra. The source of DNA for the clones was as follows; Human Immunodeficiency Virus type 1, BH10 (L. Ratner et al., Nature 312:277-284. 1985); Human Immunodeficiency Virus type 2 NIHZ (J. F. Zagury, et al., Proc. Natl. Acad. Sci. USA 85:5941-5945. 1988), Human T-cell leukemia virus type 1 pMT-2, (M. Clarke et al. Nature 305:60-62. 1983); Human T-cell leukemia virus type 2 (K. Shimotohmo et al. Proc. Natl. Acad. Sci. USA 82:3101-3105. 1985); and Human Hepatitis B Virus serotype ADW, obtained from ATCC(# 45020). Target in 50 μl of 10 mM N-2-hydroxyethelpiperazine-N′-2-ethanesulfonic acid (HEPES), 10 mM ethylenediaminetetraacetic acid (EDTA), 1% lithium lauryl sulfate, pH 7.4, was denatured at 95° C. for 5 min, cooled on wet ice, and 0.04 pmol of probe in 50 μl of 0.1 M lithium succinate buffer, pH 4.7, 2% (w/v) lithium lauryl sulfate, 1.2 M lithium chloride, 10 mM EDTA and 20 mM ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) was added. Hybridization was carried out at 60° C. for 10 min, followed by addition of 300 μl of 0.6 M sodium borate pH 8.5, 1% Triton X-100 and a second incubation at 60° C. for 6 min to hydrolyze the AE on unhybridized probe. Samples were cooled in ice water for 1 min, placed at room temperature for another 3 min, and then analyzed in a LEADER 1 luminometer equipped with automatic injection of detection reagent I (containing 0.1% hydrogen peroxide and 1 mM nitric acid) and detection reagent II (containing 1 N sodium hydroxide and a surfactant component). Some of the hybridization reactions were enhanced with the addition of 4 pmol of unlabelled “helper probe” as disclosed in Hogan et al., U.S. Pat. No. 5,030,557 entitled “Means and Methods for Enhancing Nucleic Acid Hybridization”, hereby incorporated by reference herein. An RLU value greater than 5,000 RLU was a positive result; less than 5,000 RLU was a negative result.

[0034] The following data (Table 1) show that the probes do not cross react with viral DNA from closely related viruses found in human blood or tissues. The samples also gave a positive signal when tested with a probe specific to each target, thereby confirming sample adequacy. TABLE 1 Hybridization Assay with HIV-1 probes. Probe Sequence Target: ID No. 1 2 3 4 5 6 7  1. 179,166 482 500 496 190 496 470  2. 14,992 563 394 377 409 383 465  4. 61,691 2,818 750 695 686 702 642  6. 28,038 546 408 375 356 369 372  7. 27,407 640 401 252 366 343 359  8. 45,868 1,432 395 392 386 401 404  9. 15,971 721 280 268 261 274 284 10. 59,007 714 264 280 284 272 567 11. 25,856 4,641 3,598 3,736 3,711 3,855 8,388 12. 140,691 1,846 602 694 531 534 1,236

[0035] The above data confirm that the novel probes herein disclosed and claimed are capable of distinguishing Human Immunodeficiency Virus type 1 from these viruses found in human blood.

Example 2

[0036] Amplification of HIV by PCR

[0037] To demonstrate the reactivity of the primers and probes for Human Immunodeficiency Virus type 1, the following experiment was performed. Zero, 20, or 100 copies of plasmid DNA containing Human Immunodeficiency Virus DNA was linearized with a restriction endonuclease, and added to amplification reactions containing 50 pmol of each primer, 10 mM Tris HCl pH 8, 50 mM KCl, 1.25 mM MgCl₂, 0.25 mM each of dATP, dTTP, dCTP, dGTP, and 2.5 U Tag DNA polymerase in 50 μl. The reactions were incubated at 95° C. for 1-2 min, and then cycled 35 times at 55° C. for 15 sec, 72° C. for 30 sec, and 95° C. for 20 sec in a Perkin-Elmer 9600 thermocycler or 55° C. for 30 sec, 72° C. for 60 sec, and 95° C. for 60 sec in a Perkin-Elmer 48 well thermocycler. Following cycling, the reactions were incubated at 72° C. for 6-7 min and stored at 4° C. Ten μl of the product was analyzed by hybridization protection assay with 0.04 pmol of labeled probe. The data are shown in Table 2. RLU greater than 7,000 is considered a positive result. TABLE 2 Amplification of Human Immunodeficiency Virus Type 1 by PCR Primer Probe Sequence Sequence Sample RLU ID Nos: ID. No. 0 c 20 c 100 c  19/20* 1   886 827,202 723,008  21/22* 2 2,677  24,030  48,521 *23/25 3   307 144,082 603,456 *24/27 4 4,042  81,052 163,355 *26/28 5   263 273,023 593,022 *29/30 6 1,008 328,736 366,590 *31/32 7 3,394  73,690  86,168 *33/34 8 1,648  7,152  24,027 *35/36 9   560  82,980 145,681 *39/40 10   810 279,079 299,815 *39/41 10   886 362,914 427,500  42/43* 11 5,830 680,788 130,709 # to the 5′ end of the primer. 0 c = 0 copies of HIV DNA, 20 c = 20 copies of HIV DNA, # 100 c = 100 copies of HIV DNA. Probe 1 was used in the presence of unlabeled helper probe SEQ. ID. No. 15. # Probe 7 was used in the presence of unlabeled helper probe SEQ. ID. No. 16. Probe 10 was used in the presence # of unlabeled helper probe SEQ. ID. No. 17. Probe 12 was used in the presence of unlabeled helper probe # SEQ. ID. No. 18. As the copy number increased, RLU increased. Thus, the primers of the present invention # were able to successfully amplify, by PCR, HIV type 1 target sequences which were detected using the probes of the # present invention.

Example 3

[0038] Patient samples

[0039] In this example, patient samples containing lysate prepared from 200,000 Ficoll-Hypaque purified white blood cells from individuals known to be infected with HIV type 1 or an individual not infected with HIV type 1 (negative) were analyzed as described in Example 2. These cells were prepared as described in Ryder and Kacian, entitled “Preparation of nucleic acid from blood,” U.S. Ser. No. 07/898,785, filed Jun. 12, 1992. The results are shown in Table 3. TABLE 3 PCR Assay Primer Probe Sequence Sequence Sample RLU ID Nos: ID. No. Patient 1 Patient 2 Negative  19/20* 1 27,616 71,981 886  21/22* 2 34,949 35,483 565 *23/25 3 45,230 93,529 455 *24/27 4  2,355 25,329 1,052   *26/28 5 22,345 26,014 369 *31/32 7 200,418  130,486  481 *33/34 8 43,993 40,389 705 *39/40 10 36,310 50,838 976 *39/41 10 55,582 98,504 993  42/43* 11 99,028 207,605  6,057   *44/45 12 55,082 80,388 1,496   # the primer. The primers of the present invention were able to amplify by PCR HIV type 1 target sequences present in # individuals infected with HIV. The amplified target sequences were detected by the probes of the present invention. # Thus, individuals containing HIV type 1 and an individual not containing HIV type 1 were correctly identified.

Example 4

[0040] Non-PCR Amplification

[0041] To show that the amplification oligomers also work in a transcription based amplification assay, 0, 2,000, or 20,000 copies of plasmid DNA containing HIV type 1 was linearized using a restriction endonuclease, and heated to 95° C. for two min and cooled to 37° C. for 1 min. Following addition of 800 U of MMLV reverse transcriptase the reactions were incubated for 12 min at 37° C., heated to 95° C. for two min, and cooled to 37° C. for one min. 800 U of MMLV reverse transcriptase and 400 U of T7 RNA polymerase were added and the reactions were incubated for 3 hr at 37° C. The final amplification conditions were 70 mM Tris HCl, pH 8, 35 mM KCl 15 mM KOH neutralized N-acetyl-cysteine, 6 mM rGTP, 4 mM rCTP, 4 mM rATP, 4 mM rUTP, 1 mM each of dTTP, dATP, dCTP and dGTP, and 22 mM MgCl₂ in 100 μl. Ten μl of each reaction was mixed with 40 μl of water and assayed as described for Table 1 except that the hybridization buffer contained 20 mM aldrithiol. The results in RLU are shown in Table 4. TABLE 4 Transcription-Based Amplification Assay Primer Probe Sequence Sequence RLU ID Nos: ID. No. 0 c 2,000 c 20,000 c  19/20* 1   681  24,170 190,536  21/22* 2   793  62,476 523,770 *23/25 3 2,239 812,577 1,126,045   *24/27 4 1,901 160,274 780,351 *26/28 5 2,555 877,893 1,167,756   *29/30 6   868 299,255 880,119 *31/32 7   871 129,732 969,034 *33/34 8   710 134,887 986,266 *35/36 9   884 128,981 1,021,865   *39/40 10 1,597 375,629 478,883 *39/41 10 1,264 499,304 495,509 *44/45 12 2,426  41,684 542,339 # the primer. Probe 1 was used in the presence of unlabelled helper probe SEQ. ID. No. 15. Probe 7 was used in the # presence of unlabelled helper probe SEQ. ID. No. 16, probe 10 was used in the presence of unlabelled helper probe SEQ. # ID. No. 17, and probe 12 was used in the presence of unlabelled helper probe SEQ. ID. No. 18. 0 c = 0 copies of # HIV DNA, 2,000 c = 2,000 copies of HIV DNA, 20,000 c = 20,000 copies of HIV DNA.

[0042] As the copy number increased RLU also increased. Thus, the primers of the present invention can be used to amplify HIV type 1 target sequences using a transcription based amplification assay and the amplified target sequences can be detected using the probes of the present invention.

Example 5

[0043] This example demonstrates the ability of probes for Human Immunodeficiency Virus type I to detect low levels of target oligomer produced in a transcription based amplification assay. Zero or 10 copies of plasmid DNA containing HIV type 1 sequence was linearized using a restriction endonuclease, heated in the presence of 1 μg of human DNA to 95° C. for eight minutes, and then cooled to 42° C. for six minutes. Amplification was carried out at 42° C. for two hours using 800 U MMLV reverse transcriptase and 400 U of T7 RNA polymerase, in the following reaction mix: 50 mM Tris HCl pH 8, 17.5 mM MgCl₂, 0.05 mM zinc acetate, 10% glycerol, 6.25 mM rGTP, 2.5 mM rCTP, 6.25 mM rATP, 2.5 mM rUTP, 0.2 mM dTTP, 0.2 mM dATP, 0.2 mM, 0.2 mM dCTP and 0.2 mM dGTP. Primer SEQ ID NOs. 26, 28, and 41 were used at a concentration of 30 pmol, primer SEQ ID NO. 39 was used at a concentration of 15 pmol. The entire reaction was analyzed using the hybridization protection assay with 0.04 pmol of probe in 100 μl of the hybridization buffer (supplemented with 20 mM aldrithiol) as described in Example 1. Probe SEQ ID NO. 10 was hybridized in the presence of 2 pmol unlabeled helper SEQ ID NO. 17. TABLE 5 Low Level Transcription-Based Amplification Assay RLU Primers SEQ ID NOs. Probe SEQ 0 copies 10 copies *26/28  5 1,293  64,639 *39/40 10 2,143 564,185 # sequence 5′-AATTTAATACGACTCACTATAGGGAGA-3′ attached to the 5′ end of the primer.

[0044] Other embodiments are within the following claims.

1 139 31 nucleic acid single linear 1 GACTAGCGGA GGCTAGAAGG AGAGAGATGG G 31 31 nucleic acid single linear 2 GAAGGCTTTC AGCCCAGAAG TAATACCCAT G 31 30 nucleic acid single linear 3 ATTTGCATGG CTGCTTGATG TCCCCCCACT 30 26 nucleic acid single linear 4 CTTCCCCTTG GTTCTCTCAT CTGGCC 26 37 nucleic acid single linear 5 GTCATCCATC CTATTTGTTC CTGAAGGGTA CTAGTAG 37 33 nucleic acid single linear 6 CTCCCTGACA TGCTGTCATC ATTTCTTCTA GTG 33 31 nucleic acid single linear 7 GTGGAAGCAC ATTGTACTGA TATCTAATCC C 31 37 nucleic acid single linear 8 GCTCCTCTAT TTTTGTTCTA TGCTGCCCTA TTTCTAA 37 23 nucleic acid single linear 9 CCTTTGTGTG CTGGTACCCA TGC 23 29 nucleic acid single linear 10 CTACTATTCT TTCCCCTGCA CTGTACCCC 29 29 nucleic acid single linear 11 AAAGCCTTAG GCATCTCCTA TGGCAGGAA 29 28 nucleic acid single linear 12 GCAGCTGCTT ATATGCAGGA TCTGAGGG 28 33 nucleic acid single linear 13 CAAGGCAAGC TTTATTGAGG CTTAAGCAGT GGG 33 28 nucleic acid single linear 14 ATCTCTAGCA GTGGCGCCCG AACAGGGA 28 24 nucleic acid single linear 15 TGCGAGAGCG TCAGTATTAA GCGG 24 36 nucleic acid single linear 16 CTACTTTGGA ATATTGCTGG TGATCCTTTC CATCCC 36 33 nucleic acid single linear 17 CCAATCCCCC CTTTTCTTTT AAAATTGTGG ATG 33 24 nucleic acid single linear 18 CTCGCCACTC CCCAGTCCCG CCCA 24 24 nucleic acid single linear 19 CTCGACGCAG GACTCGGCTT GCTG 24 23 nucleic acid single linear 20 CTCCCCCGCT TAATACTGAC GCT 23 31 nucleic acid single linear 21 GGCAAATGGT ACATCAGGCC ATATCACCTA G 31 25 nucleic acid single linear 22 GGGGTGGCTC CTTCTGATAA TGCTG 25 26 nucleic acid single linear 23 CAGAAGGAGC CACCCCACAA GATTTA 26 27 nucleic acid single linear 24 GACCATCAAT GAGGAAGCTG CAGAATG 27 25 nucleic acid single linear 25 CCCATTCTGC AGCTTCCTCA TTGAT 25 19 nucleic acid single linear 26 AGTGACATAG CAGGAACTA 19 26 nucleic acid single linear 27 CCATCCTATT TGTTCCTGAA GGGTAC 26 23 nucleic acid single linear 28 AGATTTCTCC TACTGGGATA GGT 23 30 nucleic acid single linear 29 GAAACCTTGT TGAGTCCAAA ATGCGAACCC 30 18 nucleic acid single linear 30 TGTGCCCTTC TTTGCCAC 18 22 nucleic acid single linear 31 CAGTACTGGA TGTGGGTGAT GC 22 35 nucleic acid single linear 32 GTCATGCTAC TTTGGAATAT TTCTGGTGAT CCTTT 35 33 nucleic acid single linear 33 CAATACATGG ATGATTTGTA TGTAGGATCT GAC 33 28 nucleic acid single linear 34 ACCAAAGGAA TGGAGGTTCT TTCTGATG 28 28 nucleic acid single linear 35 GCATTAGGAA TCATTCAAGC ACAACCAG 28 34 nucleic acid single linear 36 GCACTGACTA ATTTATCTAC TTGTTCATTT CCTC 34 38 nucleic acid single linear 37 GGGATTGGAG GAAATGAACA AGTAGATAAA TTAGTCAG 38 35 nucleic acid single linear 38 TGTGTACAAT CTAGTTGCCA TATTCCTGGA CTACA 35 22 nucleic acid single linear 39 CAAATGGCAG TATTCATCCA CA 22 23 nucleic acid single linear 40 GTTTGTATGT CTGTTGCTAT TAT 23 18 nucleic acid single linear 41 CCCTTCACCT TTCCAGAG 18 27 nucleic acid single linear 42 GAGCCCTGGA AGCATCCAGG AAGTCAG 27 21 nucleic acid single linear 43 CTTCGTCGCT GTCTCCGCTT C 21 27 nucleic acid single linear 44 CAAGGGACTT TCCGCTGGGG ACTTTCC 27 27 nucleic acid single linear 45 GTCTAACCAG AGAGACCCAG TACAGGC 27 25 nucleic acid single linear 46 GTACTGGGTC TCTCTGGTTA GACCA 25 28 nucleic acid single linear 47 CACACAACAG ACGGGCACAC ACTACTTG 28 25 nucleic acid single linear 48 CTGAGGGATC TCTAGTTACC AGAGT 25 23 nucleic acid single linear 49 CTCTGGTAAC TAGAGATCCC TCA 23 23 nucleic acid single linear 50 GTTCGGGCGC CACTGCTAGA GAT 23 23 nucleic acid single linear 51 GCAAGCCGAGT CCTGCGTCG AGA 23 27 nucleic acid single linear 52 AATTTAATAC GACTCACTAT AGGGAGA 27 31 nucleic acid single linear 53 CCCATCTCTC TCCTTCTAGC CTCCGCTAGT C 31 31 nucleic acid single linear 54 CATGGGTATT ACTTCTGGGC TGAAAGCCTT C 31 30 nucleic acid single linear 55 AGTGGGGGGA CATCAAGCAG CCATGCAAAT 30 26 nucleic acid single linear 56 GGCCAGATGA GAGAACCAAG GGGAAG 26 37 nucleic acid single linear 57 CTACTAGTAC CCTTCAGGAA CAAATAGGAT GGATGAC 37 33 nucleic acid single linear 58 CACTAGAAGA AATGATGACA GCATGTCAGG GAG 33 31 nucleic acid single linear 59 GGGATTAGAT ATCAGTACAA TGTGCTTCCA C 31 37 nucleic acid single linear 60 TTAGAAATAG GGCAGCATAG AACAAAAATA GAGGAGC 37 23 nucleic acid single linear 61 GCATGGGTAC CAGCACACAA AGG 23 29 nucleic acid single linear 62 GGGGTACAGT GCAGGGGAAA GAATAGTAG 29 29 nucleic acid single linear 63 TTCCTGCCAT AGGAGATGCC TAAGGCTTT 29 28 nucleic acid single linear 64 CCCTCAGATC CTGCATATAA GCAGCTGC 28 33 nucleic acid single linear 65 CCCACTGCTT AAGCCTCAAT AAAGCTTGCC TTG 33 28 nucleic acid single linear 66 TCCCTGTTCG GGCGCCACTG CTAGAGAT 28 31 nucleic acid single linear 67 GACUAGCGGA GGCUAGAAGG AGAGAGAUGG G 31 31 nucleic acid single linear 68 GAAGGCUUUC AGCCCAGAAG UAAUACCCAU G 31 30 nucleic acid single linear 69 AUUUGCAUGG CUGCUUGAUG UCCCCCCACU 30 26 nucleic acid single linear 70 CUUCCCCUUG GUUCUCUCAU CUGGCC 26 37 nucleic acid single linear 71 GUCAUCCAUC CUAUUUGUUC CUGAAGGGUA CUAGUAG 37 33 nucleic acid single linear 72 CUCCCUGACA UGCUGUCAUC AUUUCUUCUA GUG 33 31 nucleic acid single linear 73 GUGGAAGCAC AUUGUACUGA UAUCUAAUCC C 31 37 nucleic acid single linear 74 GCUCCUCUAU UUUUGUUCUA UGCUGCCCUA UUUCUAA 37 23 nucleic acid single linear 75 CCUUUGUGUG CUGGUACCCA UGC 23 29 nucleic acid single linear 76 CUACUAUUCU UUCCCCUGCA CUGUACCCC 29 29 nucleic acid single linear 77 AAAGCCUUAG GCAUCUCCUA UGGCAGGAA 29 28 nucleic acid single linear 78 GCAGCUGCUU AUAUGCAGGA UCUGAGGG 28 33 nucleic acid single linear 79 CAAGGCAAGC UUUAUUGAGG CUUAAGCAGU GGG 33 28 nucleic acid single linear 80 AUCUCUAGCA GUGGCGCCCG AACAGGGA 28 31 nucleic acid single linear 81 CCCAUCUCUC UCCUUCUAGC CUCCGCUAGU C 31 31 nucleic acid single linear 82 CAUGGGUAUU ACUUCUGGGC UGAAAGCCUU C 31 30 nucleic acid single linear 83 AGUGGGGGGA CAUCAAGCAG CCAUGCAAAU 30 26 nucleic acid single linear 84 GGCCAGAUGA GAGAACCAAG GGGAAG 26 37 nucleic acid single linear 85 CUACUAGUAC CCUUCAGGAA CAAAUAGGAU GGAUGAC 37 33 nucleic acid single linear 86 CACUAGAAGA AAUGAUGACA GCAUGUCAGG GAG 33 31 nucleic acid single linear 87 GGGAUUAGAU AUCAGUACAA UGUGCUUCCA C 31 37 nucleic acid single linear 88 UUAGAAAUAG GGCAGCAUAG AACAAAAAUA GAGGAGC 37 23 nucleic acid single linear 89 GCAUGGGUAC CAGCACACAA AGG 23 29 nucleic acid single linear 90 GGGGUACAGU GCAGGGGAAA GAAUAGUAG 29 29 nucleic acid single linear 91 UUCCUGCCAU AGGAGAUGCC UAAGGCUUU 29 28 nucleic acid single linear 92 CCCUCAGAUC CUGCAUAUAA GCAGCUGC 28 33 nucleic acid single linear 93 CCCACUGCUU AAGCCUCAAU AAAGCUUGCC UUG 33 28 nucleic acid single linear 94 UCCCUGUUCG GGCGCCACUG CUAGAGAU 28 24 nucleic acid single linear 95 CCGCTTAATA CTGACGCTCT CGCA 24 36 nucleic acid single linear 96 GGGATGGAAA GGATCACCAG CAATATTCCA AAGTAG 36 33 nucleic acid single linear 97 CATCCACAAT TTTAAAAGAA AAGGGGGGAT TGG 33 24 nucleic acid single linear 98 TGGGCGGGAC TGGGGAGTGG CGAG 24 24 nucleic acid single linear 99 CUCGACGCAG GACUCGGCUU GCUG 24 23 nucleic acid single linear 100 CUCCCCCGCU UAAUACUGAC GCU 23 31 nucleic acid single linear 101 GGCAAAUGGU ACAUCAGGCC AUAUCACCUA G 31 25 nucleic acid single linear 102 GGGGUGGCUC CUUCUGAUAA UGCUG 25 26 nucleic acid single linear 103 CAGAAGGAGC CACCCCACAA GAUUUA 26 27 nucleic acid single linear 104 GACCAUCAAU GAGGAAGCUG CAGAAUG 27 25 nucleic acid single linear 105 CCCAUUCUGC AGCUUCCUCA UUGAU 25 19 nucleic acid single linear 106 AGUGACAUAG CAGGAACUA 19 26 nucleic acid single linear 107 CCAUCCUAUU UGUUCCUGAA GGGUAC 26 23 nucleic acid single linear 108 AGAUUUCUCC UACUGGGAUA GGU 23 30 nucleic acid single linear 109 GAAACCUUGU UGAGUCCAAA AUGCGAACCC 30 18 nucleic acid single linear 110 UGUGCCCUUC UUUGCCAC 18 22 nucleic acid single linear 111 CAGUACUGGA UGUGGGUGAU GC 22 35 nucleic acid single linear 112 GUCAUGCUAC UUUGGAAUAU UUCUGGUGAU CCUUU 35 33 nucleic acid single linear 113 CAAUACAUGG AUGAUUUGUA UGUAGGAUCU GAC 33 28 nucleic acid single linear 114 ACCAAAGGAA UGGAGGUUCU UUCUGAUG 28 28 nucleic acid single linear 115 GCAUUAGGAA UCAUUCAAGC ACAACCAG 28 34 nucleic acid single linear 116 GCACUGACUA AUUUAUCUAC UUGUUCAUUU CCUC 34 38 nucleic acid single linear 117 GGGAUUGGAG GAAAUGAACA AGUAGAUAAA UUAGUCAG 38 35 nucleic acid single linear 118 UGUGUACAAU CUAGUUGCCA UAUUCCUGGA CUACA 35 22 nucleic acid single linear 119 CAAAUGGCAG UAUUCAUCCA CA 22 23 nucleic acid single linear 120 GUUUGUAUGU CUGUUGCUAU UAU 23 18 nucleic acid single linear 121 CCCUUCACCU UUCCAGAG 18 27 nucleic acid single linear 122 GAGCCCUGGA AGCAUCCAGG AAGUCAG 27 21 nucleic acid single linear 123 CUUCGUCGCU GUCUCCGCUU C 21 27 nucleic acid single linear 124 CAAGGGACUU UCCGCUGGGG ACUUUCC 27 27 nucleic acid single linear 125 GUCUAACCAG AGAGACCCAG UACAGGC 27 25 nucleic acid single linear 126 GUACUGGGUC UCUCUGGUUA GACCA 25 28 nucleic acid single linear 127 CACACAACAG ACGGGCACAC ACUACUUG 28 25 nucleic acid single linear 128 CUGAGGGAUC UCUAGUUACC AGAGU 25 23 nucleic acid single linear 129 CUCUGGUAAC UAGAGAUCCC UCA 23 23 nucleic acid single linear 130 GUUCGGGCGC CACUGCUAGA GAU 23 23 nucleic acid single linear 131 GCAAGCCGAG UCCUGCGUCG AGA 23 24 nucleic acid single linear 132 UGCGAGAGCG UCAGUAUUAA GCGG 24 36 nucleic acid single linear 133 CUACUUUGGA AUAUUGCUGG UGAUCCUUUC CAUCCC 36 33 nucleic acid single linear 134 CCAAUCCCCC CUUUUCUUUU AAAAUUGUGG AUG 33 24 nucleic acid single linear 135 CUCGCCACUC CCCAGUCCCG CCCA 24 24 nucleic acid single linear 136 CCGCUUAAUA CUGACGCUCU CGCA 24 36 nucleic acid single linear 137 GGGAUGGAAA GGAUCACCAG CAAUAUUCCA AAGUAG 36 33 nucleic acid single linear 138 CAUCCACAAU UUUAAAAGAA AAGGGGGGAU UGG 33 24 nucleic acid single linear 139 UGGGCGGGAC UGGGGAGUGG CGAG 24 

In the claims
 85. (New) A probe mix comprising two or more oligonucleotides, said oligonucleotides comprising a probe sequence, said sequence capable of hybridizing with HIV-1 nucleic acid sequences, said oligonucleotides further consisting of or contained within at least two nucleotide sequences selected from: (SEQ ID NO: 1) GACTAGCGGAGGCTAGAAGGAGAGAGATGGG, (SEQ ID NO: 2) GAAGGCTTTCAGCCCAGAAGTAATACCCATG, (SEQ ID NO: 3) ATTTGCATGGCTGCTTGATGTCCCCCCACT, (SEQ ID NO: 4) CTTCCCCTTGGTTCTCTCATCTGGCC, (SEQ ID NO: 5) GTCATCCATCCTATTTGTTCCTGAAGGGTACTAGTAG, (SEQ ID NO: 6) CTCCCTGACATGCTGTCATCATTTCTTCTAGTG, (SEQ ID NO: 7) GTGGAAGCACATTGTACTGATATCTAATCCC, (SEQ ID NO: 8) GCTCCTCTATTTTTGTTCTATGCTGCCCTATTTCTAA, (SEQ ID NO: 9) CCTTTGTGTGCTGGTACCCATGC (SEQ ID NO:10) CTACTATTCTTTCCCCTGCACTGTACCCC, (SEQ ID NO:11) AAAGCCTTAGGCATCTCCTATGGCAGGAA (SEQ ID NO:12) GCAGCTGCTTATATGCAGGATCTGAGGG, (SEQ ID NO:13) CAAGGCAAGCTTTATTGAGGCTTAAGCAGTGGG, (SEQ ID NO:14) ATCTCTAGCAGTGGCGCCCGAACAGGGA, (SEQ ID NO:15) TGCGAGAGCGTCAGTATTAAGCGG, (SEQ ID NO:16) CTACTTTGGAATATTGCTGGTGATCCTTTCCATCCC, (SEQ ID NO:17) CCAATCCCCCCTTTTCTTTTAAAATTGTGG ATG, (SEQ ID NO:18) CTCGCCACTCCCCAGTCCCGCCCA, (SEQ ID NO:19) CTCGACGCAGGACTCGGCTTGCTG, (SEQ ID NO:20) CTCCCCCGCTTAATACTGACGCT, (SEQ ID NO:21) GGCAAATGGTACATCAGGCCATATCACCTAG, (SEQ ID NO:22) GGGGTGGCTCCTTCTGATAATGCTG, (SEQ ID NO:23) CAGAAGGAGCCACCCCACAAGATTTA, (SEQ ID NO:24) GACCATCAATGAGGAAGCTGCAGAATG, (SEQ ID NO:25) CCCATTCTGCAGCTTCCTCATTGAT, (SEQ ID NO:26) AGTGACATAGCAGGAACTA, (SEQ ID NO:27) CCATCCTATTTGTTCCTGAAGGGTAC, (SEQ ID NO:28) AGATTTCTCCTACTGGGATAGGT, (SEQ ID NO:29) GAAACCTTGTTGAGTCCAAAATGCGAACCC, (SEQ ID NO:30) TGTGCCCTTCTTTGCCAC, (SEQ ID NO:31) CAGTACTGGATGTGGGTGATGC, (SEQ ID NO:32) GTCATGCTACTTTGGAATATTTCTGGTGATCCTTT, (SEQ ID NO:33) CAATACATGGATGATTTGTATGTAGGATCT GAC, (SEQ ID NO:34) ACCAAAGGAATGGAGGTTCTTTCTGATG, (SEQ ID NO:35) GCATTAGGAATCATTCAAGCACAACCAG, (SEQ ID NO:36) GCACTGACTAATTTATCTACTTGTTCATTTCCTC, (SEQ ID NO:37) GGGATTGGAGGAAATGAACAAGTAGATAAATTAGTCAG, (SEQ ID NO:38) TGTGTACAATCTAGTTGCCATATTCCTGGACTACA, (SEQ ID NO:39) CAAATGGCAGTATTCATCCACA, (SEQ ID NO:40) GTTTGTATGTCTGTTGCTATTAT, (SEQ ID NO:41) CCCTTCACCTTTCCAGAG, (SEQ ID NO:42) GAGCCCTGGAAGCATCCAGGAAGTCAG, (SEQ ID NO:43) CTTCGTCGCTGTCTCCGCTTC, (SEQ ID NO:44) CAAGGGACTTTCCGCTGGGGACTTTCC, (SEQ ID NO:45) GTCTAACCAGAGAGACCCAGTACAGGC, (SEQ ID NO:46) GTACTGGGTCTCTCTGGTTAGACCA, (SEQ ID NO:47) CACACAACAGACGGGCACACACTACTTG, (SEQ ID NO:48) CTGAGGGATCTCTAGTTACCAGAGT, (SEQ ID NO:49) CTCTGGTAACTAGAGATCCCTCA, (SEQ ID NO:50) GTTCGGGCGCCACTGCTAGAGAT, (SEQ ID NO:51) GCAAGCCGAGTCCTGCGTCGAGA,

oligonucleotides complementary to any of SEQ ID Nos. 1-51, and RNA equivalents and/or complements to any of SEQ ID Nos. 1-51, wherein if said probe mix comprises SEQ ID NO: 9 and SEQ ID NO:11, then said probe mix comprises at least one additional oligonucleotide not SEQ ID NO: 9 and not SEQ ID NO:11.
 86. (New) An oligonucleotide comprising a probe sequence consisting of or contained within the nucleotide sequence selected from the group consisting of: (SEQ ID NO: 3) ATTTGCATGGCTGCTTGATGTCCCCCCACT, (SEQ ID NO: 5) GTCATCCATCCTATTTGTTCCTGAAGGGTACTAGTAG, (SEQ ID NO: 6) CTCCCTGACATGCTGTCATCATTTCTTCTAGTG, (SEQ ID NO: 7) GTGGAAGCACATTGTACTGATATCTAATCCC, (SEQ ID NO: 8) GCTCCTCTATTTTTGTTCTATGCTGCCCTATTTCTAA, (SEQ ID NO:13) CAAGGCAAGCTTTATTGAGGCTTAAGCAGTGGG, (SEQ ID NO:14) ATCTCTAGCAGTGGCGCCCGAACAGGGA, (SEQ ID NO:15) TGCGAGAGCGTCAGTATTAAGCGG, (SEQ ID NO:16) CTACTTTGGAATATTGCTGGTGATCCTTTCCATCCC, (SEQ ID NO:17) CCAATCCCCCCTTTTCTTTTAAAATTGTGG ATG, (SEQ ID NO:18) CTCGCCACTCCCCAGTCCCGCCCA, (SEQ ID NO:23) CAGAAGGAGCCACCCCACAAGATTTA, (SEQ ID NO:25) CCCATTCTGCAGCTTCCTCATTGAT, (SEQ ID NO:26) AGTGACATAGCAGGAACTA, (SEQ ID NO:28) AGATTTCTCCTACTGGGATAGGT, (SEQ ID NO:29) GAAACCTTGTTGAGTCCAAAATGCGAACCC, (SEQ ID NO:30) TGTGCCCTTCTTTGCCAC, (SEQ ID NO:31) CAGTACTGGATGTGGGTGATGC, (SEQ ID NO:32) GTCATGCTACTTTGGAATATTTCTGGTGATCCTTT, (SEQ ID NO:33) CAATACATGGATGATTTGTATGTAGGATCT GAC, (SEQ ID NO:34) ACCAAAGGAATGGAGGTTCTTTCTGATG, (SEQ ID NO:35) GCATTAGGAATCATTCAAGCACAACCAG, (SEQ ID NO:36) GCACTGACTAATTTATCTACTTGTTCATTTCCTC, (SEQ ID NO:37) GGGATTGGAGGAAATGAACAAGTAGATAAATTAGTCAG, (SEQ ID NO:38) TGTGTACAATCTAGTTGCCATATTCCTGGACTACA, (SEQ ID NO:40) GTTTGTATGTCTGTTGCTATTAT, (SEQ ID NO:42) GAGCCCTGGAAGCATCCAGGAAGTCAG, (SEQ ID NO:43) CTTCGTCGCTGTCTCCGCTTC, (SEQ ID NO:46) GTACTGGGTCTCTCTGGTTAGACCA, (SEQ ID NO:47) CACACAACAGACGGGCACACACTACTTG, (SEQ ID NO:48) CTGAGGGATCTCTAGTTACCAGAGT, (SEQ ID NO:49) CTCTGGTAACTAGAGATCCCTCA, (SEQ ID NO:50) GTTCGGGCGCCACTGCTAGAGAT, (SEQ ID NO:51) GCAAGCCGAGTCCTGCGTCGAGA, (SEQ ID NO:53) CCCATCTCTCTCCTTCTAGCCTCCGCTAGTC, (SEQ ID NO:54) CATGGGTATTACTTCTGGGCTGAAAGCCTTC, (SEQ ID NO:55) AGTGGGGGGACATCAAGCAGCCATGCAAAT, (SEQ ID NO:56) GGCCAGATGAGAGAACCAAGGGGAAG, (SEQ ID NO:57) CTACTAGTACCCTTCAGGAACAAATAGGATGGATGAC, (SEQ ID NO:58) CACTAGAAGAAATGATGACAGCATGTCAGGGAG, (SEQ ID NO:59) GGGATTAGATATCAGTACAATGTGCTTCCAC, (SEQ ID NO:60) TTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAGC, (SEQ ID NO:62) GGGGTACAGTGCAGGGGAAAGAATAGTAG, (SEQ ID NO:64) CCCTCAGATCCTGCATATAAGCAGCTGC, (SEQ ID NO:65) CCCACTGCTTAAGCCTCAATAAAGCTTGCCTTG, (SEQ ID NO:66) TCCCTGTTCGGGCGCCACTGCTAGAGAT, (SEQ ID NO:67) GACUAGCGGAGGCUAGAAGGAGAGAGAUGGG, (SEQ ID NO:68) GAAGGCUUUCAGCCCAGAAGUAAUACCCAUG, (SEQ ID NO:69) AUUUGCAUGGCUGCUUGAUGUCCCCCCACU, (SEQ ID NO:70) CUUCCCCUUGGUUCUCUCAUCUGGCC, (SEQ ID NO:71) GUCAUCCAUCCUAUUUGUUCCUGAAGGGUACUAGUAG, (SEQ ID NO:72) CUCCCUGACAUGCUGUCAUCAUUUCUUCUAGUG, (SEQ ID NO:73) GUGGAAGCACAUUGUACUGAUAUCUAAUCCC, (SEQ ID NO:74) GCUCCUCUAUUUUUGUUCUAUGCUGCCCUAUUUCUAA, (SEQ ID NO:76) CUACUAUUCUUUCCCCUGCACUGUACCCC, (SEQ ID NO:78) GCAGCUGCUUAUAUGCAGGAUCUGAGGG, (SEQ ID NO:79) CAAGGCAAGCUUUAUUGAGGCUUAAGCAGUGGG, (SEQ ID NO:80) AUCUCUAGCAGUGGCGCCCGAACAGGGA, (SEQ ID NO:81) CCCAUCUCUCUCCUUCUAGCCUCCGCUAGUC, (SEQ ID NO:82) CAUGGGUAUUACUUCUGGGCUGAAAGCCUUC, (SEQ ID NO:83) AGUGGGGGGACAUCAAGCAGCCAUGCAAAU, (SEQ ID NO:84) GGCCAGAUGAGAGAACCAAGGGGAAG, (SEQ ID NO:85) CUACUAGUACCCUUCAGGAACAAAUAGGAUGGAUGAC, (SEQ ID NO:86) CACUAGAAGAAAUGAUGACAGCAUGUCAGGGAG, (SEQ ID NO:87) GGGAUUAGAUAUCAGUACAAUGUGCUUCCAC, (SEQ ID NO:88) UUAGAAAUAGGGCAGCAUAGAACAAAAAUAGAGGAGC, (SEQ ID NO:90) GGGGUACAGUGCAGGGGAAAGAAUAGUAG, (SEQ ID NO:92) CCCUCAGAUCCUGCAUAUAAGCAGCUGC, (SEQ ID NO:93) CCCACUGCUUAAGCCUCAAUAAAGCUUGCCUUG, (SEQ ID NO:94) UCCCUGUUCGGGCGCCACUGCUAGAGAU (SEQ ID NO:95) CCGCTTAATACTGACGCTCTCGCA, (SEQ ID NO:96) GGGATGGAAAGGATCACCAGCAATATTCCAAAGTAG, (SEQ ID NO:97) CATCCACAATTTTAAAAGAAAAGGGGGGATTGG, and (SEQ ID NO:98) TGGGCGGGACTGGGGAGTGGCGAG. 